The overall goal of the proposed research is to reveal how variants and homologs of bovine pancreatic ribonuclease (RNase A) promote tumor cell death. Onconase (ONC), which is an amphibian homolog of RNase A, is now undergoing Phase III human clinical trials for the treatment of malignant mesothelioma and has completed Phase II human clinical trials for the treatment of metastatic kidney cancer, refractory breast cancer, and prostate cancer. In contrast to ONC, RNase A is not cytotoxic. In the previous funding period, variants of RNase A and its human homolog were created that, like ONC, were able to evade the ribonuclease inhibitor protein (RI) that resides in the cytosol of mammalian cells. These variants were toxic to tumor cells. This finding portends the development of a new class of cancer chemotherapeutics based on mammalian ribonucleases. The Specific Aims of this proposal are (1) to enhance the antitumoral activity of RNase A by the disruption of "hot spots" identified by computational analysis of the Rl.RNase A complex, (2) to identify new cytotoxic ribonucleases from unbiased libraries using a genetic screen, (3) to reveal fundamental information about the subcellular routing of ribonucleases by using a new class of pro-fluorophores, (4) to create a ribonuclease zymogen that unleashes its cytotoxic activity in the presence of a matdx metalloprotease, and (5) to determine the three-dimensional structure of cytotoxic variants and homologs of RNase A, and thereby enhance the understanding of their antitumoral activity Significance. The proposed research will provide a detailed biochemical understanding of the antitumoral activity of ribonucleases, and could lead to new cancer chemotherapeutics based on variants and homologs of RNase A.